A widely used principle in multianalyte assays is to use labeled analyte-specific tracer molecules where concentrations of analytes in a sample can be detected on the basis of changes taking place in fluorescence-based emission signals that may be dependent on one or more excitation wavelengths used. In an ideal case, fluorescence-based emission signals related to different analytes occur on non-overlapping detection wavelength bands and therefore the emission signals can be separated from each other with straightforward optical filtering. However, in many cases, one or more of the spectra of the fluorescence-based emission signals related to different analytes is/are so wide and spectrally overlapping that it is not possible to select such detection wavelength bands that all emission signals measured on these detection wavelength bands would be free from cross-talk.
A simple principle for cross-talk correction is applicable in two-analyte assays where intensity of one of the emission signals can be measured cross-talk free using a suitable temporal detection window and/or a suitable detection wavelength band. The measured intensity of the other emission signal is corrected using the measured intensity of the first emission signal and an empirical cross-talk parameter which indicates the relation between the intensity occurring in the detection wavelength band of the first emission signal and intensity occurring in the detection wavelength band of the other emission signal in a test situation where only the analyte causing the first emission signal is present. When using this method it is worth noticing that the measured intensity of the first emission signal may be at least partially caused by a background signal that is present also when the first emission signal is not generated in detection reactions. Thus, in order to avoid too strong cross-talk correction especially in situations where the first emission signal is not generated, or generated only in a small extent, in detection reactions, the contribution of the background signal should be sufficiently eliminated from the measured intensity of the first emission signal prior to the cross-talk correction. The contribution of the background signal and the cross-talk parameter can be determined on the basis of intensities measured from appropriate test samples.
In many cases the situation is, however, more complicated so that none of the emission signals can be measured cross-talk free, i.e. the emission signals are spectrally and temporally so overlapping that it is not possible to select such temporal detection windows and/or detection wavelength bands so that at least one of the emission signals could be measured cross-talk free. Exemplifying cases where a situation of the kind described above is present are, for example, assays based on time-resolved fluorescence resonance energy transfer “TR-FRET” based multianalyte detection. Details about exemplifying FRET-based detection methods can be found for example from publications US20060147954 and V. Laitala et al., Time-resolved detection probe for homogeneous nucleic acid analyses in one-step format, Analytical Biochemistry 361 (2007) 126-131. In the method described by V. Laitala et al., each FRET-probe comprises a donor, an acceptor, and a reactive region capable of specifically hybridizing with its complementary target sequence, i.e. the target analyte to be detected with the probe under consideration. In the method described by V. Laitala et al., the time-resolved fluorescence emission signal of a population of probes of a given type comprises two signal components: a first signal component belonging to the acceptors excited due to the energy transfer from the excited donors and a second signal component belonging to the excited donors which do not participate to the energy transfer. The acceptor is preferably selected so that its emission spectrum has a maximum at a wavelength where the donor has a local minimum in its emission spectrum. The decay time of the energy transfer induced acceptor emission is dependent on the energy transfer efficiency, which in turn is inversely proportional to distance between the acceptor and the donor. Decay time of energy transfer induced acceptor emission is significantly faster when the probes are unhybridized, i.e. a short donor-acceptor distance, than when the probes are hybridized with the target analyte, i.e. a longer donor-acceptor distance. Thus, acceptor emission signal of hybridized probes can be separated from acceptor emission signal of unhybridized probes using a suitable temporal detection window. Acceptor emission signals of different probes hybridized with different target analytes can be separated from each other by using appropriate detection wavelength bands but, as mentioned above, the cross-talk complicates the situation. The situation is further complicated by the fact that the background emission caused by excited donors that do not participate to the energy transfer is dependent on the percentage of hybridized probes from all probes in a sample under consideration.